A glycosyltransferase mutant for synthesizing rebaudioside d and preparation method, product and application thereof

Abstract

The application belongs to the technical field of biological enzymes, and particularly relates to a glycosyltransferase mutant for synthesizing rebaudioside D, a preparation method, products and applications thereof. The glycosyltransferase mutant is based on the amino acid sequence shown in SEQ ID NO:1, and comprises at least one substitution mutation selected from the following positions: the 158th amino acid is mutated from S to F, or the 93rd amino acid is mutated from H to Q. The mutant can efficiently catalyze the conversion reaction from RebA to RebD, greatly accelerates the reaction process, and improves the generation rate and yield of the product. This achievement effectively overcomes the problems of low activity, low reaction efficiency and insufficient stability of the glycosyltransferase in the prior art when catalyzing the reaction, provides strong technical support for the industrialized efficient synthesis of RebD, and has important practical application value.

Description

Technical Field

[0001] This invention belongs to the field of bioenzyme technology, specifically relating to a glycosyltransferase mutant for the synthesis of rebaudioside D, its preparation method, product, and application. Background Technology

[0002] Steviol, a naturally occurring sweetener in stevia, is a diterpene glycoside commonly known as steviosides (SGs). Steviosides are a multi-component mixture characterized by high sweetness, low calories, stability, and easy solubility in water and alcohols (such as methanol and ethanol).

[0003] The main components of steviol glycosides include steviol glycosides, rebaudioside A~O, dulcitol A, stevioside, and steviol disaccharides. Among them, steviol glycosides and rebaudioside A (RA) are the most abundant, accounting for more than 80% of the total steviol glycosides, and are the main components that determine its sweetness characteristics. They are characterized by slow release of sweetness, long duration, and a taste close to sucrose; however, at high concentrations, they can exhibit a bitter taste, limiting their application range.

[0004] Recent studies have found that increasing the content of rebaudioside D (RD) can effectively improve the bitterness of steviol glycosides, while maintaining a taste similar to sucrose. Therefore, rebaudioside D is hailed as the "next-generation steviol" and its applications are becoming increasingly widespread.

[0005] Currently, the main production processes for rebaudioside D include extraction and biocatalysis. Extraction involves directly extracting, separating, and purifying rebaudioside D from stevia; however, its extremely low content in the raw material (<0.5%) limits large-scale production. Biocatalysis utilizes enzymes or microbial cells as biocatalysts to synthesize rebaudioside D. Yang Yufeng, Fei Liwen, Wang et al. expressed UGT11 in *E. coli* to achieve RebA-catalyzed synthesis of RebD, achieving a conversion rate of over 90%. Compared to extraction, this method yields higher rebaudioside D production and is therefore more widely used.

[0006] However, the UDP-glucosyltransferases used in biocatalytic methods generally suffer from low enzyme activity and low catalytic efficiency, resulting in high costs for large-scale industrial production of rebaudioside D. Therefore, it is urgent to modify this enzyme to improve the biocatalytic synthesis efficiency of rebaudioside D. Summary of the Invention

[0007] To address the aforementioned shortcomings, this invention provides a mutant of glycosyltransferase UGT11 modified by mutation technology, which improves the enzyme activity of glycosyltransferase UGT11 and enables efficient catalytic synthesis from RA to RD.

[0008] The technical solution of this invention is as follows: On one hand, the present invention provides a glycosyltransferase mutant for the synthesis of rebaudioside D, the glycosyltransferase mutant being based on the amino acid sequence shown in SEQ ID NO:1, and containing at least one substitution mutation selected from the following positions: the 158th amino acid is mutated from S to F or the 93rd amino acid is mutated from H to Q.

[0009] Specifically, the amino acid sequence of the glycosyltransferase mutant is shown in SEQ ID NO:2 or SEQ ID NO:4.

[0010] In another aspect, the present invention provides a nucleic acid encoding the aforementioned glycosyltransferase mutant, the sequence of which is as shown in SEQ ID NO:7 or SEQ ID NO:9.

[0011] In another aspect, the present invention provides an expression vector comprising the aforementioned nucleic acids.

[0012] Specifically, the expression vector is selected from plasmids, bacteriophages, viruses, or artificial chromosomes.

[0013] Preferably, the expression vector is a plasmid.

[0014] Preferably, the plasmid is selected from the pET series, pQE series, pGEX series, or pMAL series.

[0015] Preferably, the plasmid is pET-30a.

[0016] In another aspect, the present invention provides a host cell comprising the aforementioned nucleic acid or expression vector.

[0017] Specifically, the host cell can be a prokaryotic cell or a eukaryotic cell.

[0018] Preferably, the host cells include, but are not limited to, Escherichia coli, yeast, Bacillus, or Lactobacillus.

[0019] Preferably, the host cell has the accession number CGMCC No. 34492.

[0020] In another aspect, the present invention provides a method for preparing the aforementioned glycosyltransferase mutant, comprising the following steps: S1. Express the nucleic acid encoding the glycosyltransferase mutant in the host cell; S2. Culture the host cells and induce protein expression; S3. Separate and purify to obtain the glycosyltransferase mutant.

[0021] In another aspect, the present invention provides cell cultures or extracts obtained by culturing the aforementioned host cells.

[0022] In another aspect, the present invention provides an enzyme preparation comprising the aforementioned glycosyltransferase mutant, cell culture, or extract.

[0023] Specifically, the enzyme preparation also includes pharmaceutically or industrially acceptable excipients.

[0024] Preferably, the excipients include, but are not limited to, buffers, stabilizers, preservatives, or lyophilizers.

[0025] In another aspect, the present invention provides the application of the aforementioned glycosyltransferase mutants, nucleic acids, expression vectors, host cells, or enzyme preparations in the synthesis of rebaudioside D.

[0026] The beneficial effects of this invention are as follows: This invention significantly improves enzyme activity and thermostability by mutating the glycosyltransferase UGT11, enabling it to catalyze the production of RebD from RebA more efficiently, thus overcoming the problem of low activity in the prior art.

[0027] Preservation information: Biomaterial R1; Classification and nomenclature: Escherichia coli ( Escherichia coli ); Accession number: CGMCC No. 34492; Deposit date: May 9, 2025; Preservation institution: China General Microbiological Culture Collection Center, China Committee on the Preservation and Management of Microbial Culture Collections; Abbreviation of depositary institution: CGMCC; Address: No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing. Detailed Implementation

[0028] The present invention will be further clearly and completely illustrated below through embodiments. These embodiments are only some examples of the present invention and are not intended to limit the present invention, but are only for illustrating the present invention. Unless otherwise specified, the experimental methods used in the following embodiments are all conventional experiments, and the materials and reagents used in the following embodiments are commercially available unless otherwise specified.

[0029] Basic Implementation The detection methods for steviol glycosides (STV, RA, RD, RM) are as follows: HPLC was used for detection, specifically: Column: ShimNex UP C18 (4.6*150mm, 5µm); Mobile phase: A: water, B: acetonitrile; Ultraviolet detector; Detection wavelength: 210nm; Column temperature: 40℃; Flow rate: 1.0 ml / min; Drift tube temperature: 40℃; Injection volume: 10 μL; Gradient elution program: T / min (B%): 0 (20), 20 (50), 20.01 (20), 30 (20).

[0030] Elution program: 30% acetonitrile (0-30 min).

[0031] Formula for calculating molar conversion rate: Molar conversion rate = (molar amount of product generated / molar amount of initial substrate added) × 100%.

[0032] Example 1: Construction and Identification of Recombinant Escherichia coli 1. Plasmid construction Escherichia coli BL21(DE3) (Catalog No.: B528414) and DH5α competent cells (Catalog No.: A338951) were purchased from Sangon Biotech (Shanghai) Co., Ltd.; plasmid pET-30a(+) (Catalog No.: B540185) was purchased from Sangon Biotech (Shanghai) Co., Ltd.; all gene and primer synthesis and sequencing services were provided by Suzhou Genewise Biotech Co., Ltd.; 2x Phanta Flash Master Mix high-fidelity DNA polymerase (Catalog No.: P510-01) was purchased from Nanjing Novizan Biotechnology Co., Ltd.; restriction endonucleases NdeI (Code No. 1621) and XhoI (Code No. 1635) were purchased from Bio-Rad Biotechnology (Beijing) Co., Ltd. (1) Gene acquisition The UGT11 gene was synthesized by Suzhou Genewise Biotechnology Co., Ltd.

[0033] The amplification primers are shown in Table 1: Table 1 Primer sequences

[0034] Note: Italic and bold font CATATG This is the NdeI restriction site. CTCGAG This is the XhoI restriction site.

[0035] The PCR amplification program is as follows: the target fragment is amplified using 2x Phanta Flash Master Mix high-fidelity DNA polymerase. The specific reaction system and amplification program are shown in Table 2.

[0036] Table 2. PCR reaction system and amplification procedure for Phanta DNA polymerase.

[0037] The above PCR amplification products were purified and recovered using a DNA gel recovery and purification kit (Zymo Research; D4008) to obtain the purified AtSUS gene fragment.

[0038] (2) Enzyme digestion and enzyme chain The amplified DNA fragments or plasmids were digested with restriction endonucleases. In this study, QuickCut™ series restriction endonucleases were used. Digestion was performed at 37°C for 5 min, and the reaction system is shown in Table 3.

[0039] Table 3 Enzyme digestion reaction system

[0040] After purification, the digested fragments were mixed with T4 DNA ligase (Novizan; C301-01) and the purified fragments from the UGT11 gene digestion and pET30a(+) plasmid digestion, and incubated in a metal bath at 22 ℃ for 2.5 h to complete the ligation reaction. The ligation reaction system is shown in Table 4.

[0041] Table 4 Enzyme ligation reaction system

[0042] (3) Transformation verification The above enzyme-linked product was transformed (using conventional chemical transformation) into E. coli DH5α competent cells and cultured overnight. Single colonies that grew in the selection plate (50 µg / mL kanamycin) were sent to Genewiz for sequencing. After successful sequencing, the positive plasmid pET30a(+)-UGT11 was returned.

[0043] Amino acid sequence SEQ ID NO:1 UGT11: MDSGYSSSYAAAAGMHVVICPWLAFGHLLPCLDLAQRLASRGHRVSFVSTPRNISRLPPVRPALAPLVAFVALPLPRVEGLPDGAESTNDVPHDRPDMVELHRRAFDGLAAPFSEFLGTACADWVIVDVFHHWAAAAALEHKVPCAMMLLGSAHMIASIADRRLERAETESPAAAGQGRPAAAPTFEVARMKLIRTKGSSGMSLAERFSLTLSRSSLVVGRSCVEFEPETVPLLSTLRGKPITFLGLMPPLHEGRREDGEDATVRWLDAQPAKSVVYVALGSEVPLGVEKVHELALGLELAGTRFLWALRKPTGVSDADLLPAGFEERTRGRGVVATRWVPQMSILAHAAVGAFLTHCGWNSTIEGLMFGHPLIMLPIFGDQGPNARLIEAKNAGLQVARNDGDGSFDREGVAAAIRAVAVEEESSKVFQAKAKKLQEIVADMACHERYIDGFIQQLRSYKDHHHHHH* Nucleotide sequence SEQ ID NO:6 UGT11:

[0044] (4) Construction of mutant expression plasmids Based on the pET30a(+)-AtSUS plasmid, PCR amplification was performed using the amplification primers in the table. The PCR products were purified by gel excision and transformed (using conventional chemical transformation method) into E. coli DH5α competent cells and cultured overnight. Single colonies that grew in the selection plate (50 µg / mL kanamycin) were sent to Genewiz for sequencing. After successful sequencing, plasmids containing UGT11 mutant positive plasmids pET30a(+)-UGT11(S158F), pET30a(+)-UGT11(T193P), pET30a(+)-UGT11(H93Q), and pET30a(+)-UGT11(F186S) were returned.

[0045] Table 5. List of primers for mutants

[0046] Amino acid sequence SEQ ID NO:2 UGT11 (S158F): MDSGYSSSYAAAAGMHVVICPWLAFGHLLPCLDLAQRLASRGHRVSFVSTPRNISRLPPVRPALAPLVAFVALPLPRVEGLPDGAESTNDVPHDRPDMVELHRRAFDGLAAPFSEFL GTACADWVIVDVFHHWAAAAALEHKVPCAMMLLGSAHMIADRRLERAETESPAAAGQGRPAAAPTFEVARMKLIRTKGSSGMSLAERFSLTLSRSSLVVGRSCVEFEPETVPLL STLRGKPITFLGLMPPLHEGRREDGEDATVRWLDAQPAKSVVYVALGSEVPLGVEKVHELALGLELAGTRFLWALRKPTGVSDADLLPAGFEERTRGRGVVATRWVPQMSILAHAAV GAFLTHCGWNSTIEGLMFGHPLIMLPIFGDQGPNARLIEAKNAGLQVARNDGDGSFDREGVAAAIRAVAVEEESSKVFQAKAKKLQEIVADMACHERYIDGFIQQLRSYKDHHHHHH* Amino acid sequence SEQ ID NO:3 UGT11 (T196P): MDSGYSSSYAAAAGMHVVICPWLAFGHLLPCLDLAQRLASRGHRVSFVSTPRNISRLPPVRPALAPLVAFVALPLPRVEGLPDGAESTNDVPHDRPDMVELHRRAFDGLAAPFSEFLGTACADWVIVDVFHHWAAAAALEHKVPCAMMLLGSAHMIASIADRRLERAETESPAAAGQGRPAAAPTFEVARMKLIRPKGSSGMSLAERFSLTLSRSSLVVGRSCVEFEPETVPLLSTLRGKPITFLGLMPPLHEGRREDGEDATVRWLDAQPAKSVVYVALGSEVPLGVEKVHELALGLELAGTRFLWALRKPTGVSDADLLPAGFEERTRGRGVVATRWVPQMSILAHAAVGAFLTHCGWNSTIEGLMFGHPLIMLPIFGDQGPNARLIEAKNAGLQVARNDGDGSFDREGVAAAIRAVAVEEESSKVFQAKAKKLQEIVADMACHERYIDGFIQQLRSYKDHHHHHH* Amino acid sequence SEQ ID NO:4 UGT11 (H93Q): MDSGYSSSYAAAAGMHVVICPWLAFGHLLPCLDLAQRLASRGHRVSFVSTPRNISRLPPVRPALAPLVAFVALPLPRVEGLPDGAESTNDVPQDRPDMVELHRRAFDGLAAPFSEFLGTACADWVIVDVFHHWAAAAALEHKVPCAMMLLGSAHMIASIADRRLERAETESPAAAGQGRPAAAPTFEVARMKLIRTKGSSGMSLAERFSLTLSRSSLVVGRSCVEFEPETVPLLSTLRGKPITFLGLMPPLHEGRREDGEDATVRWLDAQPAKSVVYVALGSEVPLGVEKVHELALGLELAGTRFLWALRKPTGVSDADLLPAGFEERTRGRGVVATRWVPQMSILAHAAVGAFLTHCGWNSTIEGLMFGHPLIMLPIFGDQGPNARLIEAKNAGLQVARNDGDGSFDREGVAAAIRAVAVEEESSKVFQAKAKKLQEIVADMACHERYIDGFIQQLRSYKDHHHHHH* Amino acid sequence SEQ ID NO:5 UGT11 (F186S): MDSGYSSSYAAAAGMHVVICPWLAFGHLLPCLDLAQRLASRGHRVSFVSTPRNISRLPPVRPALAPLVAFVALPLPRVEGLPDGAESTNDVPHDRPDMVELHRRAFDGLAAPFSEFLGTACADWVIVDVFHHWAAAAALEHKVPCAMMLLGSAHMIASIADRRLERAETESPAAAGQGRPAAAPTSEVARMKLIRTKGSSGMSLAERFSLTLSRSSLVVGRSCVEFEPETVPLLSTLRGKPITFLGLMPPLHEGRREDGEDATVRWLDAQPAKSVVYVALGSEVPLGVEKVHELALGLELAGTRFLWALRKPTGVSDADLLPAGFEERTRGRGVVATRWVPQMSILAHAAVGAFLTHCGWNSTIEGLMFGHPLIMLPIFGDQGPNARLIEAKNAGLQVARNDGDGSFDREGVAAAIRAVAVEEESSKVFQAKAKKLQEIVADMACHERYIDGFIQQLRSYKDHHHHHH* Nucleotide sequence SEQ ID NO:7 UGT11 (S158F): Nucleotide sequence SEQ ID NO:8 UGT11 (T196P): Nucleotide sequence SEQ ID NO:9 UGT11 (H93Q): Nucleotide sequence SEQ ID NO:10 UGT11 (F186S):

[0047] 1.5 Construction of Recombinant Strains The constructed plasmid was transformed (using conventional chemical transformation) into competent Escherichia coli BL21(DE3) cells and cultured overnight. Validation primers (upstream primer test-pET-F (SEQ ID NO:11): 5'-CATCGGTGATGTCGGCGATATAG -3', downstream primer test-pET-R (SEQ ID NO:12): 5'-CCGGATATAGTTCCTCCTTTCAGCA -3') were designed to verify single colonies grown in a screening plate (50 µg / mL kanamycin) using colony PCR, thus constructing the corresponding recombinant strain.

[0048] Example 2: Induction and expression of recombinant Escherichia coli and preparation of purified enzyme solution 1. Induced expression Select a single colony of the engineered bacteria and inoculate it into 2YT medium containing 50 µg / mL kanamycin (2YT medium formula: 16 g / L tryptone, 10 g / L yeast extract, 5 g / L sodium chloride) and incubate at 37℃ and 220 rpm for 6 h. Transfer the culture to 150 ml of 2YT medium containing 50 µg / mL kanamycin at a 1% (v / v) inoculation rate and incubate at 37℃ and 220 rpm until the OD600 reaches 0.6-0.8. Add IPTG to a final concentration of 0.3 mM and incubate at 16℃ for 20 h to induce expression. Centrifuge at 4℃ and 6000 rpm for 15 min, discard the supernatant, and keep the precipitate for later use.

[0049] 2. Preparation of crude enzyme solution A 10% (m / v) UGT11 mutant bacterial culture was prepared using Tris (50 mM pH 7.5) solution. The bacterial cells were homogenized by high pressure (900 bar) to release the target protein. The culture was centrifuged at 4°C and 6000 rpm for 15 min. The supernatant enzyme solution was collected to obtain crude UGT11 mutant enzyme solution for later use.

[0050] 3. Protein purification (1) Nickel gravity column pretreatment: HisSep Ni-NTAAgarose Resin was loaded into a suitable purification column by gravity, the column was rinsed with 2 column volumes of deionized water, and the column was equilibrated with 2 column volumes of Tris. (2) Sample loading: Add the crude enzyme solution prepared above to the purification column dropwise, paying attention to controlling the loading rate to ensure that the target protein and Ni are mixed. 2+ Sufficient contact is necessary to improve purification yield; (3) Washing: Washing a 2-cylinder volume of Wash Buffer; (4) Elution: Elute with 30 mL of Elution Buffer and collect the eluent, which is the target protein solution; (5) Column regeneration: one column volume of 1M imidazole washing, followed by two column volumes of deionized water rinsing. (6) Storage: Finally, store the resin in deionized water at 4°C; (7) Ultrafiltration: Add 50mM Tris buffer (pH7.5) to the ultrafiltration tube, centrifuge at 5000rpm and 4℃ for 10min, and discard the centrifuged liquid; add an appropriate amount of the eluted target protein solution, centrifuge at 5000rpm and 4℃ to a volume of about 1mL; add an equal volume of 50mM Tris buffer (pH7.5), centrifuge at 5000rpm and 4℃ to a volume of about 1mL, and repeat this step once to remove high concentrations of imidazole and obtain the protein purification solution.

[0051] Example 3: Enzyme activity assay of recombinant Escherichia coli (UGT11 mutant) UGT11 mutant (RA→RD) enzyme activity assay: Definition: The amount of enzyme required to generate 1 µmol RD in 1 minute at a reaction temperature of 37℃ is defined as 1 enzyme activity unit (U).

[0052] (1) Enzyme-catalyzed reaction 1 mM UDPG, 1 mM RA, 50 mM Tris (pH 7.5), and 0.1 mg / mL purified protein were added to a 200 μL reaction system in a 96-well plate. The reaction was carried out at 37℃ for 30 min. After adding an equal volume of methanol to the reaction solution, the mixture was centrifuged and filtered through a 0.22 μm filter membrane. The amount of RD generated was detected by liquid chromatography.

[0053] (2) Enzyme activity calculation Specific activity (U / g) = RD generation (μmol) ÷ reaction time (min) ÷ protein mass (g) The detection results, obtained using the enzyme activity assay method described above, are shown in Table 6. Table 6

[0054] As shown in Table 6, the specific activity of the glycosyltransferase UGT11 mutant UGT11 (S158F) induced by recombinant Escherichia coli was significantly increased to 403.47 U / g, which is 9.89 times higher than that of UGT11.

[0055] Example 4: Determination of the thermal stability of enzyme activity in recombinant Escherichia coli (UGT11 mutant) After incubating the above mutants in a buffer system at 55°C for 30 min, the enzyme activity was measured according to the method described in Example 3. The results are shown in Table 7. Table 7

[0056] As shown in Table 7, the AtSUS mutant AtSUS (P94N) induced by recombinant Escherichia coli retained 93.66% of its enzyme activity after incubation at 55℃ for 30 min, while the wild-type enzyme activity retained 42.89% under the same treatment conditions. The strain expressing this mutant UGT11 (S158F) was named R1 (CGMCC No. 34492).

[0057] Example 5: Whole-cell catalytic synthesis of RebD Wet cells of engineered bacterium R1 (UGT11(S158F)) were obtained according to the induction expression method described in Example 2, and wet cells of AtSUS expression were prepared using the same method. In a reaction vessel, UDP to a final concentration of 0.1 mmol / L, sucrose to a final concentration of 0.5 M, and Reb A to a final concentration of 20 g / L were added sequentially. The volume was adjusted to 100 mL with 50 mmol / L Tris-HCl buffer (pH 7.5). 100 U of UGT11(S158F) and AtSUS expression bacteria wet cells were added. The mixture was stirred at 220 rpm for 12 hours. After the reaction, 100 μL of the reaction solution was diluted with 900 μL of anhydrous methanol, centrifuged at 10,000 rpm for 3 min at 4℃, and the supernatant was filtered through a membrane and analyzed by HPLC. The results showed that Reb A was almost completely converted, and the Reb D conversion rate was 96.49%.

[0058] The above detailed description is a specific illustration of one feasible embodiment of the present invention, and this embodiment is not intended to limit the patent scope of the present invention. It should be noted that all equivalent implementations or modifications made without departing from the present invention should be included within the scope of the technical solution of the present invention. Therefore, the protection scope of the present invention should be determined by the appended claims.

Claims

1. A glycosyltransferase mutant for the synthesis of rebaudioside D, characterized in that, The amino acid sequence of the glycosyltransferase mutant is shown in SEQ ID NO:2 or SEQ ID NO:

4.

2. A nucleic acid encoding the glycosyltransferase mutant of claim 1, characterized in that, The sequence of the nucleic acid is as shown in SEQ ID NO:7 or SEQ ID NO:

9.

3. An expression vector comprising the nucleic acid of claim 2.

4. The expression vector according to claim 3, characterized in that, The expression vector is selected from plasmids, viruses, or artificial chromosomes.

5. The expression vector according to claim 4, characterized in that, The expression vector is a plasmid.

6. The expression vector according to claim 5, characterized in that, The plasmids are selected from the pET series, pQE series, pGEX series, or pMAL series.

7. The expression vector according to claim 5, characterized in that, The plasmid is pET-30a.

8. A host cell comprising the nucleic acid of claim 2 or the expression vector of any one of claims 3-7.

9. The host cell according to claim 8, characterized in that, The host cell is a prokaryotic cell or a eukaryotic cell.

10. The host cell according to claim 9, characterized in that, The host cell is Escherichia coli, yeast, Bacillus, or Lactobacillus.

11. The host cell according to claim 10, characterized in that, The host cell has the accession number CGMCC No. 34492.

12. A method for preparing the glycosyltransferase mutant of claim 1, characterized in that, Includes the following steps: S1. Express the nucleic acid encoding the mutant in the host cell; S2. Culture the host cells and induce protein expression; S3. Separate and purify to obtain the glycosyltransferase mutant.

13. An enzyme preparation, characterized in that, Including the glycosyltransferase mutant of claim 1.

14. The use of the glycosyltransferase mutant of claim 1, the nucleic acid of claim 2, the expression vector of any one of claims 3-7, the host cell of any one of claims 8-11, or the enzyme preparation of claim 13 in the synthesis of rebaudioside D.

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